Thermostable glutaminase and thermostable glutaminase gene

ABSTRACT

A completely novel glutaminase is provided: (a) a protein consisting of an amino acid sequence represented by SEQ ID NO: 2, or (b) a protein consisting of an amino acid sequence derived from the amino acid sequence represented by SEQ ID NO: 2 by deletion, substitution or addition of one or more amino acids, and possessing glutaminase activity. This protein is a novel glutaminase possessing excellent thermostability and the like.

FIELD OF THE INVENTION

[0001] The present invention relates to a glutaminase, a glutaminasegene, a recombinant DNA and a method for producing the glutaminase.

BACKGROUND OF THE INVENTION

[0002] Glutaminase (L-Glutamine amidohydrolase, EC3.5.1.2, hereinafter,referred to as glutaminase) is an enzyme that hydrolyzes glutamine intoglutamic acid and ammonia. Glutaminase is known to play an importantrole in the food-processing industry, particularly when food flavorings,such as soy sauce, that is obtained by enzymatic degradation of protein,is produced. When soy sauce is produced, a protein, as a raw material,is degraded into peptides and finally into constitutive amino acids bythe action of various proteinases produced by Aspergillus. Glutamicacid, which is a kind of constitutive amino acid, plays a central roleamong flavor-components of soy sauce. Two pathways have been devised togenerate glutamic acid in soy sauce manufacture. One pathway is thedirect generation of glutamic acid in the above described degradationprocess of the protein raw material (1st pathway) and the other pathwayis the generation of glutamic acid by converting the glutamine generatedin the degradation process of the protein raw material into glutamicacid by the action of glutaminase (2nd pathway). Storage proteins ofplants including soya beans that are the raw materials in soy saucemanufacture are rich in acidic amino acid, such as glutamic acid andasparatic acid. Most acidic amino acid is known to exist in the form ofamide, such as glutamine and asparagine. In addition, glutaminegenerated by the degradation of raw materials in the soy sauce brewingprocess changes non-enzymatically and relatively quickly into tastelesspyroglutamic acid. Therefore, in soy sauce brewing, primary importancehas been placed on the reaction which converts glutamine into glutamicacid (the 2nd pathway).

[0003] In soy sauce manufacture, the use of yellow koji molds (e.g.Aspergillus sojae) capable of high production of glutaminase has beenshown to cause increases in the amount of glutamic acid in soy saucemoromi (soy sauce moromi is formed by treating the raw materials for soysauce so as to be ready for fermentation) (see Yamamoto et. al. J.Ferment. Technol., Vol. 52, No. 8, 564-569 (1974)), and that within thissoy sauce moromi, there exists a correlation between glutaminase of theinsoluble fractions (also described as fractions of cell surface andcell first-surface, intracellular fractions, fractions of microbial bodysurface and fractions within the microbial body) of yellow koji moldsand glutamic acid (see Nippon Shoyu Kenkyusho Zasshi Vol.5, No.1, 21-25,1979). Thus, the glutaminase produced by koji molds has been recognizedas important.

[0004] Aspergillus (including yellow koji mold), Aspergillus oryzae andAspergillus sojae, has traditionally been used in the production ofbrewed food in Japan, such as miso, soy sauce and sake from old times.These microbes are particularly important industrially, because of theirhigh productivity of enzymes and high reliability of safety as attestedby their long-standing use.

[0005] Some of the glutaminases produced by the microbes of the genusAspergillus including these yellow koji molds have been purified andtheir properties have been reported. Intra- and extra-microbialglutaminases have also been purified from Aspergillus oryzae and theirproperties have been studied (see Yano, T et al, J. Ferment. Technol.,Vol.66, No.2, 137-143 (1988)). All of these glutaminases have molecularweights of approximately 113,000 and they all have similar properties.Further, 2 types of extra-microbial glutaminases, which are differentfrom the above types, have also been purified from Aspergillus oryzae,and their properties have been studied (see WO99/60104, and JP PatentPublication (Unexamined Application) No. 2002-218986). The genes ofthese extra-microbial glutaminases have also been isolated and analyzed.Further, extramicrobial glutaminases have also been purified fromAspergillus sojae and their genes have also been reported (see JP PatentPublication (Unexamined Application) No. 2000-166547). Furthermore, aglutaminase gene which is different from the above genes has also beenisolated from Aspergillus sojae (see Japanese Patent Application No.2001-187433).

[0006] The localization of the glutaminase of yellow koji molds has beenstudied. Each fraction of 3 broadly classified locations: extracellular,cell surface and intracellular (cell membrane and cytoplasm) fractionshas been studied for their properties and their distribution ratiosusing crude enzyme solutions. As a result, it has been reported thatmost glutaminases are located in the cell surface and intracellularly(see Nippon Shoyu Kenkyusho Zasshi Vol.11, No.3, 109-114, 1985).

[0007] Glutaminases derived from yellow koji molds and glutaminase genesthat have been shown so far are all extra-microbial glutaminases. Basedon analysis of molecular weight and enzymatic properties, however, ithas been suggested that the only intra-microbial glutaminase as obtainedby Yano et al. may be identical to extra-microbial glutaminase. Noglutaminase and genes thereof shown to be located definitely in the cellsurface and intracellularly have been reported. As described above,insoluble glutaminase is known to have an effect in soy saucemanufacture, so that isolation of these glutaminases is stronglydesired. Further, the use of these glutaminases has also been limited interms of industrial use, because of their low quantity of production andthe like. Hence, isolation of the gene that enables mass-preparation ofsuch glutaminases is also strongly desired.

[0008] Among the production methods for food flavorings which involvesenzymatic degradation of proteins, an extremely effective and efficientmethod for efficiently producing food flavorings having rich glutamicacid content involves the degradation of proteins at high temperatures,because it enhances the degradation rate of protein and preventscontamination by saprophytes. However, as the temperature rises, sincethe efficiency of glutamine to pyroglutamic acid conversion alsoincreases, glutaminase must quickly act under such high temperatures.Therefore, high optimal temperature and thermostability are required forglutaminase to be utilizable under such conditions. Hence, isolation ofglutaminase having these properties has been strongly desired.

SUMMARY OF THE INVENTION

[0009] Consequently, an object of the present invention is to provide acompletely novel glutaminase, a glutaminase gene, a recombinant DNA anda method for producing the glutaminase, in particular, to provide aglutaminase with excellent thermastability, a gene of the glutaminasewith excellent thermostability, a recombinant DNA and a method forproducing the glutaminase with excellent thermostability.

[0010] As a result of intensive studies to attain the above objectives,we have completed the present invention by succeeding in cloning a novelglutaminase gene from yellow koji molds:

[0011] That is, the present invention is:

[0012] 1. A protein, which is the following (a) or (b):

[0013] (a) a protein consisting of an amino acid sequence represented bySEQ ID NO: 2;

[0014] (b) a protein consisting of an amino acid sequence derived fromthe amino acid sequence represented by SEQ ID NO: 2 by deletion,substitution or addition of one or more amino acids, and possessingglutaminase activity.

[0015] 2. A protein or a partial fragment thereof, which consists of anamino acid sequence having 70% or more sequence homology with the fulllength amino acid sequence represented by SEQ ID NO: 2, and possessesglutaminase activity.

[0016] 3. A glutaminase gene, which encodes the following protein (a) or(b):

[0017] (a) a protein consisting of the amino acid sequence representedby SEQ ID NO: 2;

[0018] (b) a protein consisting of an amino acid sequence derived fromthe amino acid sequence represented by SEQ ID NO: 2 by deletion,substitution or addition of one or more amino acids, and possessingglutaminase activity.

[0019] 4. A glutaminase gene, which encodes a protein or a partialfragment thereof consisting of an amino acid sequence having 70% or moresequence homology with the full length amino acid sequence representedby SEQ ID NO: 2, and possesses glutaminase activity.

[0020] 5. A glutaminase gene, which consists of the following DNA (a) or(b);

[0021] (a) a DNA consisting of a nucleotide sequence represented by SEQID NO: 1;

[0022] (b) a DNA hybridizing under stringent conditions to a DNAcomprising a nucleotide sequence that is complementary to the DNAconsisting of the nucleotide sequence represented by SEQ ID NO: 1, andencoding a protein possessing glutaminase activity.

[0023] 6. A recombinant DNA, wherein the gene of the above 3, 4 or 5 isinserted to a vector DNA.

[0024] 7. A transformant or a transductant, which comprises therecombinant DNA of the above 6.

[0025] 8. A method for producing glutaminase, which comprises culturingthe transformant or transductant of the above 7 in a medium, andcollecting glutaminase from the culture product.

[0026] In addition, in the present invention, the nucleotide sequencerepresented by SEQ ID NO: 1 and the amino acid sequence represented bySEQ ID NO: 2 are identical to SEQ ID NOS: 13493 and 13494, respectively,in the specification of prior application No. 2001-403261 (filing date:Dec. 27, 2001).

[0027] The present invention will be described in detail as follows.

[0028] To clone a glutaminase gene or a gene that contains the gene andencodes a protein possessing glutaminase (enzyme) activity (hereinafter,may also be simply referred to as glutaminase gene), we searched thegenome sequence database of a yellow koji mold, Aspergillus oryzaestrain RIB40, so as to identify a gene having a nucleotide sequencediffering from that of the conventionally known glutaminase gene. Yellowkoji molds were cultured in various media and the gene expression wasstudied. Thus, we found that the gene was expressed in microbial bodiescultured in bran media, and then cloned cDNA from RNA extracted from themicrobial bodies. The full length ORF of the obtained cDNA was insertedinto a yeast expression plasmid vector (pYES2.1/V5-His-TOPO), and thenthe functions were analyzed using yeast (INVSc) which were transformedby the plasmid. As a result, this cDNA was confirmed to encodeglutaminase, and we succeeded in mass-producing glutaminase derived fromthe gene product.

[0029] 1. Glutaminase of the Present Invention

[0030] The glutaminase of the present invention is a protein comprisingthe amino acid sequence represented by SEQ ID NO: 2. For example, theenzyme can be purified from the culture product of yellow koji molds,such as Aspergillus sojae or Aspergillus oryzae. In addition, the enzymecan be obtained by allowing the glutaminase gene that has been clonedfrom the above yellow koji molds and the like to be expressed in anappropriate host vector system.

[0031] The glutaminase of the present invention may comprise an aminoacid sequence derived from the amino acid sequence represented by SEQ IDNO: 2 by deletion, substitution or addition of one or more amino acids,as long as it possesses enzyme activity. Here, the term “(one or) moreamino acids” means normally 2 to 300 amino acids, preferably 2 to 170amino acids, more preferably 2 to 50 amino acids, and most preferably 2to 10 amino acids. The number of amino acids varies depending on thepositions or types of amino acid residues in the three-dimensionalstructure of a glutaminase protein. Further, as long as the proteinpossesses glutaminase activity, it may be a protein or a partialfragment thereof which comprises an amino acid sequence showing 70% ormore, preferably 75% or more, more preferably 80% or more, and mostpreferably 85% or more sequence homology with the full length amino acidsequence represented by SEQ ID NO: 2.

[0032] To determine sequence homology between the two amino acidsequences or nucleotide sequences, sequences are pre-treated to reach anoptimum state for comparison. For example, the alignment with onesequence is optimized by inserting a gap into the other sequence.Afterwards, amino acid residues or nucleotides of each site arecompared. When an amino acid residue or a nucleotide at a site of thefirst sequence is identical to an amino acid residue or a nucleotide ata corresponding site of the second sequence, these sequences areidentical to each other at that site. Sequence homology between twosequences is expressed as a percentage of the number of sites identicalbetween sequences to the total number of sites (all the amino acids ornucleotides).

[0033] According to the above principle, sequence homology between twoamino acid sequences or nucleotide sequences is determined by thealgorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA87:2264-2268, 1990 and Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993).The BLAST program using such an algorithm was developed by Altschul etal (J. Mol. Biol. 215:403-410, 1990). Further, the Gapped BLAST is aprogram with higher sensitivity to determine sequence homology comparedto the BLAST (Nucleic Acids Res. 25:3389-3402, 1997). The above programis mainly used to search databases for sequences showing high sequencehomology with a given sequence. For example, these programs areavailable on the website of the National Center for BiotechnologyInformation (USA) on the Internet.

[0034] To express sequence homology between sequences, values determinedusing BLAST 2 Sequences software (FEMS Microbiol Lett., 174:247-250,1999) developed by Tatiana A. Tatusova et al., are used in the presentspecification. This software is available and also obtainable on thewebsite of the National Center for Biotechnology Information (USA) onthe Internet. Programs and parameters to be used herein are as follows.In the case of amino acid sequences, the blastp program is used, andOpen gap: 11 and extension gap: 1 penalties, gap x#dropoff: 50, expect:10, word size: 3 and Filter: ON are used as parameters. In the case ofnucleotide sequences, the blastn program is used, and Reward for amatch: 1, Penalty for a mismatch: −2, Strand option: Both strands, Opengap: 5 and extension gap: 2 penalties, gap x#dropoff: 50, expect: 10,word size: 11 and Filter: ON are used as parameters. All of theseparameters are used as default values on the web site.

[0035] However, when a sequence showing significant sequence homology isnot found using the above BLAST software, a sequence showing sequencehomology can be further searched from databases using the highsensitivity FASTA software (W. R. Pearson and D. J. Lipman, Proc. Natl.Acad. Sci., 85:2444-2448, 1988). FASTA software can be used, forexample, on the web site of the genome net. In this case, default valuesare also used as parameters. For example, when a search is preformed fora nucleotide sequence, nr-nt is used as a database and the ktup value inthis case is 6. In any cases, when a 30% or more, 50% or more, or 70% ormore overlap of the whole is not shown, functional correlation cannot benecessarily be assumed. Thus, such values are not used as values showingsequence homology between two sequences.

[0036] The above respective methods are used to search for a sequenceshowing sequence homology mainly from databases. In the presentinvention, homology analysis can be employed using Genetyx Win Ver.5(Genetyx) as a means of determining sequence homology of individualsequences. This method is based on the Lipman-Pearson method (Science,227: 1435-1441, 1985) which is frequently used as a high-speed andhigh-sensitivity method. When sequence homology of nucleotide sequencesis analyzed, regions encoding proteins (CDS or ORF) are used, ifpossible. Unit Size to compare=2 and Pick up Location=5 are entered asparameters, and results are indicated with %. The sequence homology ofan alignment showing the highest point is used as a result. When a 30%or more, 50% or more, or 70% or more overlap with a query sequence isnot shown, functional correlation is not necessarily assumed. Thus, theresult is not used as a value showing sequence homology between twosequences. For example, even if there is a completely matched region ofabout several nucleotides/residues, this may likely be a mere randomchance result. Moreover, in case of a match of several % in length ofthe whole, even when a particularly functional motif is contained, it isdifficult to determine that the two sequences exhibits the same functionas with the whole.

[0037] Specifically, when homology search is performed for the aminoacid sequence represented by SEQ ID NO: 2 using each of the abovemethods, the highest sequence homology among those of known glutaminasesis about 36% of a glutaminase (derived from Cryptococcus nodaensis, JPPatent Publication (Unexamined Application) No. 2002-262887). Thus, aglutaminase comprising the amino acid sequence represented by SEQ ID NO:2 can be said to be completely novel.

[0038] 2. Cloning of Glutaminase Gene

[0039] The glutaminase gene of the present invention can be obtainedfrom, for example, yellow koji molds, such as Aspergillus sojae orAspergillus oryzae, other filamentous fungi, or other fungi. Morespecifically, an example is Aspergillus sojae strain RIB40 (Aspergillussojae var. viridis Murakami, anamorph; ATCC42149). Total RNA iscollected by conventional techniques from the culture product obtainedby culturing these microbes cultured in media under conditions forproducing glutaminase. An example of a medium that can be used herein isa bran medium prepared by adding 2.22 g of deionized water to 2.78 g ofwheat bran, and then auto-claved at 121° C. for 50 minutes. Afterculturing in the above medium for an appropriate time, for example, for30 hours, an appropriate amount of the culture product (for example, 1g) is transferred into a mortar filled with liquid nitrogen, crushedwith a pestle, and then total RNA is prepared by the Cathala et al'smethod. (DNA, 2 (4): 329-335, 1983).

[0040] RT-PCR is performed using the thus obtained total RNA as atemplate. As primers, any combination of primers may be employed, aslong as it allows the amplification of the glutaminase gene of thepresent invention. For example, oligonucleotides having sequences of SEQID NO: 7 and SEQ ID NO: 8, respectively, can be used. For example,RT-PCR can be performed by conventional methods using a commerciallyavailable kit, such as a RNA LA-PCR Kit (TAKARA SHUZO). The resultingDNA containing the glutaminase gene of the present invention can beinserted into, for example, a plasmid vector, such as pCR2.1-TOPO orpYES2.1/V5-His-TOPO (both manufactured by Invitrogen). The nucleotidesequence of the thus obtained DNA can be determined by Sanger's methodusing a commercially available reagent and a DNA sequencer. Examples ofthe thus obtained DNA containing the glutaminase gene of the presentinvention and the amino acid sequence of the glutaminase encoded by thisgene are exemplified in SEQ ID NO: 1 (SEQ ID NO: 13493 of priorapplication No. 2001-403261) and SEQ ID NO: 2 (SEQ ID NO: 13494 of priorapplication No. 2001-403261), respectively.

[0041] In addition to the above genes, the glutaminase gene of thepresent invention may be a gene encoding a protein which comprises anamino acid sequence derived from the amino acid sequence represented bySEQ ID NO: 2 by deletion, substitution or addition of one or more aminoacids, as long as the protein possesses glutaminase activity. Inaddition to selection by hybridization to be described later, such agene can also be obtained by various known method for introducingmutations.

[0042] The glutaminase gene of the present invention can also beobtained by a selection method using hybridization, as described below.Examples of a gene source include yellow koji molds, such as Aspergillussojae or Aspergillus sojae. From these organisms, RNA or genomic DNA isprepared by conventional methods, and then inserted into a plasmid orphage, thereby preparing libraries. Next, nucleic acids to be used asprobes are labeled by any method according to the detection method to beused. A nucleic acid that may be used as a probe has a length with whichsufficient specificity can be obtained. For example, such a nucleic acidcontains a part of or the whole sequence of SEQ ID NO: 1, that is, itcontains at least 100 bases or more, preferably 200 bases or more, morepreferably 450 bases or more, and most preferably 700 bases or more ofthe sequence of SEQ ID NO: 1. Subsequently, clones that hybridize understringent conditions to labeled probes are selected from the abovelibraries. Regarding hybridization, when plasmid libraries are used,colony hybridization can be performed, and when phage libraries areused, plaque hybridization can be performed. The term “stringentconditions” means conditions wherein signals caused by specific hybridsare clearly distinguishable from that of non-specific hybrids. Thestringent conditions vary depending on the hybridization system, thetype, sequence and length of probes to be used herein. Such conditionscan be determined by changing the temperature for hybridization andvarying the temperature for washing and salt concentrations. Forexample, when the signals caused by non-specific hybrids are alsostrongly detected, specificity can be enhanced by elevating thetemperature for hybridization and washing, and if necessary, loweringthe salt concentration for washing. In addition, when the signals causedby specific hybrids are not also detected, hybrids can be stabilized bylowering the temperature for hybridization and washing, and ifnecessary, increasing the salt concentration for washing. Such anoptimization can be easily performed by researchers in this technicalfield.

[0043] Under specific examples of the stringent conditions,hybridization is performed overnight (8 to 16 hours) using 5×SSC,blocking agent (Boehringer Mannheim) for 1.0% (W/V) nucleic acidhybridization, 0.1% (W/V) N-lauroyl sarcosine and 0.02% (W/V) SDS, andthen washing is performed twice, for 15 minutes each, using 0.5×SSC and0.1% (W/V) SDS, and preferably, using 0.1×SSC and 0.1% (W/V) SDS. Thetemperature for hybridization and washing is 52° C. or more, preferably57° C. or more, more preferably 62° C. or more, and most preferably 67°C. or more.

[0044] Further, a nucleotide sequence is considered to encode a proteinpossessing activity substantially equivalent to that of the glutaminaseof the present invention, when it shows 75% or more, preferably 80% ormore, further preferably 85% or more, and most preferably 90% or moresequence homology with a 500 nucleotide or more part of or the wholenucleotide sequence of SEQ ID NO: 1.

[0045] DNA showing the above-described sequence homology in thenucleotide sequence, or showing that in the coding amino acid sequencecan be obtained using hybridization as an indicator as described above.Alternatively, for example, such DNA can also be found easily from DNAgroups having unknown functions that have been obtained by genomicnucleotide sequence analysis or the like or from public databases, forexample by a search using the above BLAST software. Such search methodsare normally employed by researchers in this technical field.

[0046] That the thus obtained DNA encodes a protein possessingglutaminase activity can be confirmed by, to be described later in “5.Production of glutaminase,” incorporating DNA into an appropriatevector, transforming an appropriate host, culturing the transformant,and then measuring glutaminase activity.

[0047] 3. Construction of Recombinant Vector

[0048] The recombinant vector of the present invention can be obtainedby ligating the glutaminase gene of the present invention onto anappropriate vector. Any vector that allows the production of glutaminasein a host to be transformed can be used. For example, plasmids, cosmids,phages, viruses, chromosome incorporation type vectors and artificialchromosome vectors can be used.

[0049] A marker gene may also be contained in the above vector to enableselection of transformed cells. Examples of a marker gene include genes,such as URA3 and niaD, which complement the auxotrophy of hosts, andresistance genes against drugs, such as ampicillin, kanamycin oroligomycin. In addition, the recombinant vector preferably contains apromoter sequence which allows expression of the gene of the presentinvention in a host cell, or other regulation sequences (for example, anenhancer sequence, terminator sequence and polyadenylation sequence).Specific examples of such a promoter include the GAL1 promoter, the amyBpromoter and the lac promoter. Further, a tag may also be added toenable purification. For example, a linker sequence is properlyconnected downstream of a glutaminase gene, and then 6 or more codons ofnucleotide sequences encoding histidines are connected, thereby enablingpurification through a nickel column.

[0050] 4. Obtainment of Transformant

[0051] The transformant of the present invention can be obtained bytransforming a host with the recombinant vector of the presentinvention. Examples of a host are not specifically limited, as long asthey can produce the glutaminase of the present invention, and includeyeast such as Saccharomyces cerevisiae, and Zygosaccharomyces rouxii,filamentous fungi, such as Aspergillus sojae, Aspergillus oryzae andAspergillus niger, and bacteria, such as Escherichia coli and Bacillussubtilis. Transformation can be performed by any known method dependingon the host to be used herein. In the case of yeast, for example, amethod using lithium acetate (Methods Mol. Cell. Biol., 5, 255-269(1995)) can be used. In the case of filamentous fungi, for example, amethod using polyethylene glycol and calcium chloride (Mol. Gen. Genet.,218, 99-104 (1989)) after protoplast preparation can be used. Whenbacteria are used, for example, a method using electroporation (MethodsEnzymol., 194, 182-187(1990)) can be used.

[0052] 5. Production of Glutaminase

[0053] A method for producing the glutaminase of the present inventioncomprises culturing the transformant or the transductant of the presentinvention, and collecting glutaminase protein from the resulting cultureproduct. A medium and a culturing method may be appropriately selectedaccording to host type and a regulatory sequence for expression in arecombinant vector. For example, when the host is Saccharomycescerevisiae and a regulatory sequence for expression is the GAL 1promoter, for example, microbes pre-cultured in a liquid minimal mediumcontaining raffinose as a carbon source are diluted, inoculated andcultured in a liquid minimal medium containing galactose and raffinoseas carbon sources, so as to allow the cells to produce the glutaminaseof the present invention. Further, for example, when the host isAspergillus sojae and a regulatory sequence for expression is the amyBpromoter, for example, the cells are cultured in a liquid minimal mediumcontaining maltose as a carbon source, so as to allow the cells toproduce the glutaminase of the present invention.

[0054] Moreover, when the host is Escherichia coli and a regulatorysequence for expression is the lac promoter, for example, the cells arecultured in a liquid medium containing IPTG, so that the glutaminase ofthe present invention can be produced. When the glutaminase of thepresent invention is produced within the bacteria or on the bacterialsurface, the bacteria are separated from the medium and thenappropriately treated, so that the glutaminase of the present inventioncan be obtained. For example, when the glutaminase is produced on themicrobial surface of Saccharomyces cerevisiae, the microbial body itselfis used as an enzyme agent so as to disrupt the microbial bodies, andnon-ionic surfactant such as Triton X-100, Tween-20 or Nonidet P-40 isallowed to act at a low concentration. Centrifugation is then performed,and then the glutaminase of the present invention can be collected fromthe supernatant. When the glutaminase of the present invention isproduced in the culture solution, the microbial bodies are removed bycentrifugation, filtration or the like, so that the glutaminase of thepresent invention can be obtained. In any cases, the glutaminase of thepresent invention having a higher purity can be obtained by anyconventional method using an ammonium sulfate fraction, various types ofchromatography, alcohol precipitation, ultrafiltration or the like.

[0055] Examples of a method for measuring the titer of glutaminaseinclude [measurement method 1] which involves quantitatively determiningL-glutamic acid generated by hydrolysis of L-glutamine, and [measurementmethod 2] which involves quantitatively determining ammonia generated byenzyme reaction. A commercial kit, such as F-kit ammonia (RocheDiagnosis) may be employed for the [measurement method 2]. [Measurementmethod 1] was employed as a method for measuring the titer of thisenzyme.

[0056] Specifically, 500 μl of 0.2 M phosphate buffer (pH 7.0) and 250μl of an enzyme solution were added to 250 μl of 2% (W/V) L-glutaminesolution, the solution was allowed to react at 37° C. for 20 minutes,and then 250 μl of 0.75 N perchloric acid solution was added to thesolution to stop reaction. Then, 125 μl of 1.5 N sodium hydroxidesolution was added to the solution, thereby neutralizing the reactionsolution. The above reaction solution was centrifuged (10,000 r.p.m., 10minutes). To 100 μl of the supernatant, 1.0 ml of 0.1 M hydroxylaminehydrochloride buffer solution (pH 8.0) containing 50 mM EDTA.Na, 1.0 mlof 20 mM NAD+solution (Oriental Yeast) and 50 μl of 500 unit/mlL-glutamate dehydrogenase solution (SIGMA) were added. Then, thesolution was allowed to react at 37° C. for 30 minutes, and thenabsorbance at 340 nm was measured using a spectral photometer. With apreviously prepared calibration curve of L-glutamic acid, whereby theproduction amount had been previously checked, and the amount of enzymethat generates 1 μ mole of glutamic acid per minute under the aboveconditions is defined as 1 unit (U).

[0057] In particular, the glutaminase activity of the glutaminase of thepresent invention is superior to all known glutaminases under hightemperature conditions. Specifically, though the conversion efficiencyfrom glutamine to pyroglutamic acid increases as temperature rises, theuse of the glutaminase of the present invention enables rapid hydrolysisof glutamine into glutamic acid under high temperature. Therefore, theuse of the glutaminase of the present invention enables enzyme reactionunder high temperature conditions during the production process for foodor the like, and prevents contamination by saprophytes, so that foodflavorings which are rich in glutamic acid content can be efficientlyproduced.

BRIEF DESCRIPTION OF THE DRAWINGS

[0058]FIG. 1 is a characteristic figure showing the results of examiningthe thermostability of this enzyme.

[0059]FIG. 2 is a characteristic figure showing the result of examiningthe optimal temperature of this enzyme.

BEST MODE FOR CARRYING OUT THE INVENTION

[0060] The present invention will be described more specifically by thefollowing examples. However, the present invention is not limited bythese examples.

[0061] 1. Cloning of glutaminase gene of Aspergillus sojae

[0062] Approximately 100,000 conidiospores of Aspergillus sojae strainRIB40 (Aspergillus sojae var. viridis Murakami, anamorph; ATCC42149)were inoculated into 5 g of a bran medium (described above) in a 150 mlErlenmeyer flask, and then statically cultured at 30° C. for 30 hours,thereby obtaining the culture product. The culture product was put intoa mortar that had been previously cooled by pouring liquid nitrogen intothe mortar. After further adding liquid nitrogen into the mortar, theculture product was thoroughly crushed with a pestle that had beenpre-cooled in liquid nitrogen. Total RNA was extracted from the crushedmicrobes by the Cathala et al's method [DNA, 2 (4): 329-335, 1983].Furthermore, DNase I treatment was performed using a DNA-free kit(Ambion) so that contaminating DNA was degraded. RT-PCR was performedusing 1.0 μg of the obtained total RNA, as a template, and Marathon cDNAAmplification Kit (Clontech).

[0063] Primers used for reverse transcription reaction were thoseattached to the kit and having an adapter sequence on the 3′ side ofoligo dT. Reverse transcription reaction was performed at 42° C. for 60minutes.

[0064] Subsequently, according to the instructions attached to the kit,a cocktail containing RNaseH, DNA polymerase, DNA ligase and the likewas added to the above reverse transcription reaction product, and thenthe mixture was allowed to react at 16° C. for 90 minutes. T4 DNApolymerase was further added to the mixture, and then the mixture wasallowed to react at 16° C. for 50 minutes, thereby synthesizing adouble-stranded cDNA library.

[0065] Next, adapter DNA, DNA ligase and the like were added to theabove reaction product, so that a double-stranded cDNA library having anadapter bound thereto was synthesized. Compositions of the reactionsolutions and reaction conditions were respectively employed accordingto the attached instructions.

[0066] Next, primers were prepared in reference to the genome databaseof Aspergillus sojae. Then, 5′RACE and 3′RACE were performed using thedouble-stranded cDNA library obtained above as a template and FirstChoice RLM-RACE Kit (Ambion) and Marathon cDNA Amplification Kit(Clontech). As a result, approximately 400 bp and 700 bp amplificationfragments were respectively obtained, so that expression of the gene ofthe enzyme was confirmed. In addition, a primer of SEQ ID NO: 3 and thatof SEQ ID NO: 4 were used for 5′RACE, and a primer of SEQ ID NO: 5 andthat of SEQ ID NO: 6 were used for 3′RACE. After the transcriptioninitiation point and the transcription termination point were clarifiedusing the RACE method, primers of SEQ ID NO: 7 and SEQ ID NO: 8 weredesigned. Using the double-stranded cDNA library obtained above as atemplate, PCR amplification was performed from a position immediatelybefore the initiation codon on the 5′ side, and to a positionimmediately before the termination codon on the 3′ side. Takara EX TaqDNA Polymerase (TAKARA SHUZO) was used as a thermostable DNA polymerase,and the composition of the reaction solution was prepared according tothe instructions attached to the polymerase.

[0067] After reaction at 94° C. for 2 minutes, the PCR reaction wasperformed for 30 cycles, each cycle consisting of 94° C. for 30 seconds,55° C. for 30 seconds, and 72° C. for 2 minutes. Then, another reactionwas performed at 72° C. for 5 minutes. When a part of the amplificationproduct was subjected to 0.7% agarose gel electrophoresis, anapproximately 1.8 kb band was confirmed. In addition, GeneAmp 5700Sequence detection system (PE Applied Biosystems) was used as a thermalcycler, and the temperature was controlled by a calculate controlmethod.

[0068] Next, the above amplification product was inserted intopYES2.1V5-His-TOPO vector using pYES2.1TOPO TA Expression Kit(Invitrogen), and then into Escherichia coli strain TOP10F′ (Invitrogen)was transformed, thereby obtaining transformants. Plasmids wereextracted from the transformants using QlAprep spin Miniprep Kit(QIAGEN). The obtained plasmids were treated using restriction enzymes,Xba I and Hind III (both produced by TAKARA SHUZO), so that thedirection of the gene insertion was confirmed. Sequence reaction wasperformed using CEQ™ DTCS-Quick Start Kit (BECKMAN COULTER), and thenthe nucleotide sequence was determined using CEQ2000XL sequencer(BECKMAN COULTER).

[0069] As a result, DNA sequence of a 1884 bases open reading frame(ORF) represented by SEQ ID NO: 1 was shown. This plasmid “pYESAsgahB”was deposited under FERM BP-8260 on Dec. 12, 2002 to the NationalInstitute of Advanced Industrial Science and Technology, InternationalPatent Organism Depository (Chuo-6, 1-1-1, Higashi, Tsukuba-shi,Ibaraki, Japan). In addition, the nucleotide sequence ranging from theinitiation codon to a position immediately before the termination codonof a clone contained in pYESAsgahB is shown in SEQ ID NO: 1. Theanalysis of the above nucleotide sequence revealed that the DNA encodesa protein comprising 628 amino acid residues. This amino acid sequenceis shown in SEQ ID NO: 2.

[0070] Furthermore, a sequence having high sequence homology with thisamino acid sequence was searched over known amino acid sequencedatabase. NCBI blastp (http://www.ncbi.nlm.nih.gov/BLAST/) was used forthis search, and nr was designated as a database. As a result, nomatched sequence was found, and the sequence having the highest sequencehomology was ORF near MEL (GenBank: CAA85738), which was a protein,having unknown functions, of the yeast Saccharomyces cerevisiae. Thesequence homology of the full length coding region was approximately 42%as examined with analytical software GENETYX-WIN Ver. 5.0. In addition,when NCBI blastp was used, no known glutaminase was found to have highhomology with the amino acid sequence represented by SEQ ID NO: 2.

[0071] Further, a sequence having high sequence homology with thenucleotide sequence represented by SEQ ID NO: 1 was searched. NCBIblastn (http://www.ncbi.nlm.nih.gov/BLAST/) was used for this search,and nr was designated as a database. As a result, no matched sequencewas found. The sequence homology of the full length coding region of theprotein ORF, near MEL (GenBank: CAA85738), having unknown functions, ofSaccharomyces cerevisiae, which showed the highest sequence homologywith the amino acid sequence as described above, was 50% and thesequence contained over 1884 bases in length as examined by theanalytical software GENETYX-WIN Ver. 5.0. In addition, when the NCBIblastn was used, no known glutaminase gene was found to have highhomology with the nucleotide sequence represented by SEQ ID NO: 1.

[0072] 2. Expression of Glutaminase cDNA

[0073] With the above plasmid pYESAsgahB, the protein of interest(glutaminase) can be expressed by induction using galactose. INVSc1(Invitrogen, Genotype: MATa, his3 Δ 1, leu2, trp1-289, ura3-52/MATα,his3 Δ 1, leu2, trp1-289, ura3-52) was used as a host. The host yeastwas transformed by the lithium acetate method using the above plasmidpYESAsgahB. 0.67% Yeast Nitrogenbase without amino acids (Difco), 2%raffinose (Wako Pure Chemical Industries), and 0.192% Yeast SyntheticDrop-out Medium Supplement Without uracil (SIGMA) were used for aselection medium. The lithium acetate method was performed according to“A protein experimental protocol-functional analysis-” (P63-P88 CellTechnology, separate volume: SHUJUNSHA).

[0074] Next, using the obtained transformant, protein was expressedaccording to the protocols attached to pYES2.1TOPO TA Expression Kit.The transformants were inoculated from colonies in 20 ml of theselection medium in a 200 ml baffle sided Erlenmeyer flask, and then thetransformants were cultured with shake at 30° C. for about 14 hours at140 rpm, thereby preparing a seed culture. The turbidity (OD₆₀₀) of theseed culture was measured, and then the seed culture was inoculated intoa medium to induce protein expression to have an initial turbidity ofOD₆₀₀=0.4. A 500 ml Sakaguchi flask was used for culturing in the mediumto induce protein expression, and culturing with shake was performed in50 ml of the medium at 30° C. at 140 rpm. The medium to induce proteinexpression used herein was a selection medium containing 1% raffinoseand 2% galactose (Wako Pure Chemical Industries) as carbon sources.

[0075] At 30 hours after the start of induction, the culture solutionwas centrifuged at 3000 rpm for 10 minutes, thereby obtaining thesupernatant as a fraction of extra-microbial secretory protein and theprecipitation as a fraction of the microbes. An extraction buffer in anequivalent volume with the pellet (20 mM Tris-HCI (pH7.5), 1 mM EDTA, 5mM MgCl₂, 50 mM KCl, 5% glycerol, 3 mM DTT, 1% protease inhibitormix/DMSO solution (Wako Pure Chemical Industries)) was added to thefraction of the microbes for suspension, thereby preparing a suspensionsolution of the microbes. An equivalent volume of glass beads was addedto the suspension solution, the solution was vigorously agitated using amulti-beads shocker for 30 seconds. Then, the solution was cooled in icefor 1 minute and 30 seconds. After repeating this procedure for 15times, centrifugation at 15000 rpm was performed for 20 minutes, therebyobtaining the supernatant as a fraction of intra-microbial solubleprotein and the precipitation as a fraction of the residues of microbes.

[0076] Glutaminase activity was measured using the fraction ofintra-microbial soluble protein obtained as a crude enzyme solution asdescribed above. Table 1 shows the results. Numerical values in Table 1denote glutaminase activity (mU/mg) per total amount of intra-microbialsoluble protein at 30 hours after induction of protein expression.“Vector” denotes the transformant of plasmid pYES2.1-V5-his-TOPO,“AsgahB” denotes the transformant of plasmid pYESAsgahB, respectively.In addition, (−) indicates that culturing was performed in a medium thatcontained no galactose and was not for inducing protein expression, and(+) indicates that culturing was performed in a medium that containedgalactose and was for inducing protein expression. TABLE 1 Vector (−)Vector (+) AsgahB (−) AsgahB (+) 6.1 5.1 5.1 123.8

[0077] The results shown in Table 1 revealed that the transformants ofplasmid pYESAsgahB cultured in the medium for inducing proteinexpression showed glutaminase activity approximately 24-fold greaterthan that of the transformants of plasmid pYES2.1-V5-his-TOPO. Further,it was also revealed that the transformants of plasmid pYESAsgahB showedglutaminase activity approximately 24-fold greater than that ofculturing in the medium that contained no galactose and was not forinducing protein expression. As described above, it was confirmed thatthe gene obtained by the present invention was glutaminase gene, and itbecame clear that the use of this gene enables mass-production ofglutaminase.

[0078] 3. Optimum Temperature and Thermostability of Glutaminase

[0079] When pYES2.1TOPO TA Expression Kit is used, V5 epitope tag andthen 6×His tag are added to the C-terminal site of this enzyme. Enzymewas purified from a crude enzyme solution of intra-microbial solublefractions obtained in a manner similar to the method of the above 2using TALON™ Purification Kit (Clontech). However, 1×Elution/Wash bufferattached to the Kit was used instead of the extraction buffer to preparethe crude enzyme solution of the intra-microbial soluble fractions. Thepurification was performed by a method according to the attachedprotocols. Specifically, resin that had been previously equilibratedwith 1×Elution/Wash buffer was added to the obtained crude enzymesolution, and then the solution was gently agitated at 4° C. for 30minutes to allow the enzyme to be adsorbed to the resin. The resin withthe enzyme adsorbed thereto was centrifuged at 1000 rpm for 5 minutes,thereby separating into the resin and the supernatant to be used as anon-adsorption fraction. The glutaminase activity of the crude enzymesolution and that of the non-adsorption fraction were measured, therebyconfirming that the enzyme was adsorbed to the resin. Subsequently, theresin with the enzyme adsorbed thereto was washed with a 10-fold volumeof 1×Elution/Wash buffer, and then centrifuged at 1000 rpm for 5minutes, thereby collecting the resin. After this procedure was repeatedtwice, the resin was suspended in 1-fold volume of 1×Elution/Washbuffer, and then the suspension was transferred into a gravity-flowcolumn. After the resin was washed with a 5-fold volume of1×Elution/Wash buffer, elution was performed with a 5-fold volume of1×Elution buffer, so that an active fraction was collected. With theprocedures as described above, purified glutaminase was obtained.

[0080] The thermostability and optimum temperature of the above purifiedenzyme were examined.

[0081] (I) Thermostability (heat-stability)

[0082] The enzyme was subjected to heat treatment at 30 to 60° C. for 10minutes or 30 minutes in 0.1 M phosphate buffer (pH 7.0), and thencooled sufficiently. Then, the activity was measured in 0.1 M phosphatebuffer (pH 7.0) at 37° C. The reactivity of a sample that had not beensubjected to heat treatment for 30 minutes was set as 100%, and thereactivity at each temperature was obtained as a relative value. FIG. 1shows the results. The results shown in FIG. 1 revealed that the enzymeshowed 80% or more remaining activity at 50° C. or less.

[0083] (II) Optimum Temperature

[0084] The activity was measured in 0.1 M phosphate buffer (pH 7.0) at30 to 60° C. With the highest value as 100%, the reactivity at eachtemperature was obtained as a relative value. FIG. 2 shows the results.The results shown in FIG. 2 revealed that the optimum temperature of theenzyme was 55° C.

[0085] Table 2 summarizes the optimum temperatures (FIG. 2) andthermostabilities (FIG. 1) of known glutaminases derived from kojimolds, Aspergillus sojae and Aspergillus sojae. Temperature described inthe row “thermostability” in Table 2 denotes the temperature of heattreatment at which 80% or more remaining activity was shown when heattreatment was performed for a duration parenthesed. TABLE 2 JP Patent JPPatent Publication Publication (Unexamined (Unexamined Application)Application) The enzyme Yano, T. et al W099/60104 No. 2000-166547 No.2002-218986 Origin Aspergillus Aspergillus Aspergillus AspergillusAspergillus oryzae oryzae oryzae sojae sojae Optimum 55 37-45 45 50 45temperature (° C.) Thermostability 50° C. or less 37° C. or less 45° C.or less 45° C. or less 40° C. or less (Duration of (30 minutes) (10minutes) (10 minutes) (30 minutes) (30 minutes) heat treatment)

[0086] The results shown in Table 2 revealed that the enzyme has a highoptimum temperature and excellent thermostability compared toconventionally known glutaminases derived from the genus Aspergillus.

[0087] Industrial Applicability

[0088] According to the present invention, there are provided acompletely novel protein, glutaminase gene, recombinant DNA and a methodfor producing the glutaminase. According to the present invention, itbecame possible to improve the above glutaminase by protein engineering.The present invention can also be used for producing enzyme for foodprocessing, and improving microorganisms to be used for producing brewedfoods.

[0089] Sequence Listing Free Text

[0090] SEQ ID NOS: 3 to 8 are primers.

1 8 1 1884 DNA Aspergillus oryzae RIB40 1 atgctctcct ctgttctccttgtcctcttc tcagcggccg tgggggcaaa gaccgtgccc 60 aacggccaaa cactcaccctcaacggcata ccctactatc tcagtgggat cccgatttcg 120 aatttctcac ataatgttttcgataaagca agcaacgatg tcgatatctt cccttttaca 180 gtcatccaga cttctagcaccgttcatagt agctttctga gtgaaacagt tgccaatttt 240 acccagcagg atgacgtgttccaacctgcg tttcttcaga ccgtctattt gacttcttct 300 gttgaggcgt cgcaaatcgacgaactgtct ggcagcgaag ctttgcacca gtttgacaat 360 aagatgttcc taaccgaatctgatgcttcc ttgtctacgc cccttccgaa tggaccttat 420 tttgcttccg cccgcacaggacacattttc agagcctatc gtctctactc tgatgactct 480 ttggcgttca tctcggccgctattagcgat gagagtggtg gtttcattcc tatgactgga 540 gttacagagg gcgtcatgacgaaaaatgtc gctgtcccat cccgtctcta ctacacccct 600 actgctgaaa agcctttagctggtttccgg ctggccgtga aggatatatt ccacattaag 660 ggtcttagaa ccagtggtggaagccgtgcg tactactacc tctacgatga gcagaatgtt 720 accactccct ctgtgcaacggctctttgac ctgggtgccg taatggtcgg aaaggtgggc 780 actgttcaat tcgccaatggtgatcgtcct actgcggact gggtcgatct gcactgtcca 840 ttcaaccccc gaggggacggatatcaatat cccagtggct cctcgtctgg ttcgggtgct 900 gccatcgccg cgtatgaatggttagatctg gctattggca gtgacacggg tggttccatg 960 cgcggacctg ccggtgtgcagggtatctat ggtaatcggc catcaactgg ggctatcact 1020 ttagagcatg ccttgccactctcgcctcca ctcgatacag ccggtatgtt cgcacgaagc 1080 gcgtctttat ggtcaaagaccgtccaagcc tggtacccca acttcaaccg cagctatcca 1140 tcccacccca aacagctctacctctctcac agcaactggg acgagtccac cgcacccgaa 1200 gcaaacgaac atctggaaacattcatgcag agactcgaag atttcctgga tacaaatcgc 1260 acaatcgtca acgtcacagaacgttggtcc gaaacccaca actcaccctc tttgatcaac 1320 ctcctgaaca caacctacgcctacctagtc ggcgtcggcc aatggaataa tctcgccaaa 1380 ggcttcttcg cagactacgcccaatcccac gacggccgtc gcccattcat caatcccggt 1440 cccttggccc gctgggaatggggccaagca aacggtggaa acgcatccta cgacgccgcc 1500 ctgcataaca tgactgtcttccgagactgg tggtcgacgt ccggatacgg acgttctgat 1560 gatgattctt gctcggaaggtatcttcgta cacgcctggg ccaccggagc agcagactac 1620 cgtaaccggt acttcaaccctcctggtccc ccgttcggat tcacagacga cgctatcgcc 1680 gttttcgcgg gcgcgcctgaagttgttgtc ccattgggcg agtcgcctta taacagtacc 1740 atcacgttgc acgaggagtatctccctgtt tcgatcggct tgcagatggc tagaggctgc 1800 gatcgggcac ttgctgagttggtggatgat ctaggcaagg cagggatttt gaagcctgtt 1860 tctgcgggct cgagattatattct 1884 2 628 PRT Aspergillus oryzae RIB40 2 Met Leu Ser Ser Val LeuLeu Val Leu Phe Ser Ala Ala Val Gly Ala 1 5 10 15 Lys Thr Val Pro AsnGly Gln Thr Leu Thr Leu Asn Gly Ile Pro Tyr 20 25 30 Tyr Leu Ser Gly IlePro Ile Ser Asn Phe Ser His Asn Val Phe Asp 35 40 45 Lys Ala Ser Asn AspVal Asp Ile Phe Pro Phe Thr Val Ile Gln Thr 50 55 60 Ser Ser Thr Val HisSer Ser Phe Leu Ser Glu Thr Val Ala Asn Phe 65 70 75 80 Thr Gln Gln AspAsp Val Phe Gln Pro Ala Phe Leu Gln Thr Val Tyr 85 90 95 Leu Thr Ser SerVal Glu Ala Ser Gln Ile Asp Glu Leu Ser Gly Ser 100 105 110 Glu Ala LeuHis Gln Phe Asp Asn Lys Met Phe Leu Thr Glu Ser Asp 115 120 125 Ala SerLeu Ser Thr Pro Leu Pro Asn Gly Pro Tyr Phe Ala Ser Ala 130 135 140 ArgThr Gly His Ile Phe Arg Ala Tyr Arg Leu Tyr Ser Asp Asp Ser 145 150 155160 Leu Ala Phe Ile Ser Ala Ala Ile Ser Asp Glu Ser Gly Gly Phe Ile 165170 175 Pro Met Thr Gly Val Thr Glu Gly Val Met Thr Lys Asn Val Ala Val180 185 190 Pro Ser Arg Leu Tyr Tyr Thr Pro Thr Ala Glu Lys Pro Leu AlaGly 195 200 205 Phe Arg Leu Ala Val Lys Asp Ile Phe His Ile Lys Gly LeuArg Thr 210 215 220 Ser Gly Gly Ser Arg Ala Tyr Tyr Tyr Leu Tyr Asp GluGln Asn Val 225 230 235 240 Thr Thr Pro Ser Val Gln Arg Leu Phe Asp LeuGly Ala Val Met Val 245 250 255 Gly Lys Val Gly Thr Val Gln Phe Ala AsnGly Asp Arg Pro Thr Ala 260 265 270 Asp Trp Val Asp Leu His Cys Pro PheAsn Pro Arg Gly Asp Gly Tyr 275 280 285 Gln Tyr Pro Ser Gly Ser Ser SerGly Ser Gly Ala Ala Ile Ala Ala 290 295 300 Tyr Glu Trp Leu Asp Leu AlaIle Gly Ser Asp Thr Gly Gly Ser Met 305 310 315 320 Arg Gly Pro Ala GlyVal Gln Gly Ile Tyr Gly Asn Arg Pro Ser Thr 325 330 335 Gly Ala Ile ThrLeu Glu His Ala Leu Pro Leu Ser Pro Pro Leu Asp 340 345 350 Thr Ala GlyMet Phe Ala Arg Ser Ala Ser Leu Trp Ser Lys Thr Val 355 360 365 Gln AlaTrp Tyr Pro Asn Phe Asn Arg Ser Tyr Pro Ser His Pro Lys 370 375 380 GlnLeu Tyr Leu Ser His Ser Asn Trp Asp Glu Ser Thr Ala Pro Glu 385 390 395400 Ala Asn Glu His Leu Glu Thr Phe Met Gln Arg Leu Glu Asp Phe Leu 405410 415 Asp Thr Asn Arg Thr Ile Val Asn Val Thr Glu Arg Trp Ser Glu Thr420 425 430 His Asn Ser Pro Ser Leu Ile Asn Leu Leu Asn Thr Thr Tyr AlaTyr 435 440 445 Leu Val Gly Val Gly Gln Trp Asn Asn Leu Ala Lys Gly PhePhe Ala 450 455 460 Asp Tyr Ala Gln Ser His Asp Gly Arg Arg Pro Phe IleAsn Pro Gly 465 470 475 480 Pro Leu Ala Arg Trp Glu Trp Gly Gln Ala AsnGly Gly Asn Ala Ser 485 490 495 Tyr Asp Ala Ala Leu His Asn Met Thr ValPhe Arg Asp Trp Trp Ser 500 505 510 Thr Ser Gly Tyr Gly Arg Ser Asp AspAsp Ser Cys Ser Glu Gly Ile 515 520 525 Phe Val His Ala Trp Ala Thr GlyAla Ala Asp Tyr Arg Asn Arg Tyr 530 535 540 Phe Asn Pro Pro Gly Pro ProPhe Gly Phe Thr Asp Asp Ala Ile Ala 545 550 555 560 Val Phe Ala Gly AlaPro Glu Val Val Val Pro Leu Gly Glu Ser Pro 565 570 575 Tyr Asn Ser ThrIle Thr Leu His Glu Glu Tyr Leu Pro Val Ser Ile 580 585 590 Gly Leu GlnMet Ala Arg Gly Cys Asp Arg Ala Leu Ala Glu Leu Val 595 600 605 Asp AspLeu Gly Lys Ala Gly Ile Leu Lys Pro Val Ser Ala Gly Ser 610 615 620 ArgLeu Tyr Ser 625 3 30 DNA Artificial Sequence Description of ArtificialSequencePrimer DNA 3 tctgaaaatg tgtcctgtgc gggcggaagc 30 4 30 DNAArtificial Sequence Description of Artificial SequencePrimer DNA 4attgtcaaac tggtgcaaag cttcgctgcc 30 5 34 DNA Artificial SequenceDescription of Artificial SequencePrimer DNA 5 aaagagctca aaatgctctcctctgttctc cttg 34 6 26 DNA Artificial Sequence Description ofArtificial SequencePrimer DNA 6 tctcacagca actgggacg agtccac 26 7 34 DNAArtificial Sequence Description of Artificial SequencePrimer DNA 7aaagagctca aaatgctctc ctctgttctc cttg 34 8 30 DNA Artificial SequenceDescription of Artificial SequencePrimer DNA 8 agaatataat ctcgagcccgcagaaacagg 30

1. A protein, which is the following (a) or (b): (a) a proteinconsisting of an amino acid sequence represented by SEQ ID NO: 2; (b) aprotein consisting of an amino acid sequence derived from the amino acidsequence represented by SEQ ID NO: 2 by deletion, substitution oraddition of one or more amino acids, and possessing glutaminaseactivity.
 2. A protein or a partial fragment thereof, which consists ofan amino acid sequence having 70% or more sequence homology with thefull length amino acid sequence represented by SEQ ID NO: 2, andpossesses glutaminase activity.
 3. A glutaminase gene, which encodes thefollowing protein (a) or (b): (a) a protein consisting of the amino acidsequence represented by SEQ ID NO: 2; (b) a protein consisting of anamino acid sequence derived from the amino acid sequence represented bySEQ ID NO: 2 by deletion, substitution or addition of one or more aminoacids, and possessing glutaminase activity.
 4. A glutaminase gene, whichencodes a protein or a partial fragment thereof consisting of an aminoacid sequence having 70% or more sequence homology with the full lengthamino acid sequence represented by SEQ ID NO: 2, and possessesglutaminase activity.
 5. A glutaminase gene, which consists of thefollowing DNA (a) or (b); (a) a DNA consisting of a nucleotide sequencerepresented by SEQ ID NO: 1; (b) a DNA hybridizing under stringentconditions to a DNA consisting of a nucleotide sequence that iscomplementary to the DNA comprising the nucleotide sequence representedby SEQ ID NO: 1, and encoding a protein possessing glutaminase activity.6. A recombinant DNA, wherein the gene of the above 3, 4 or 5 isinserted to a vector DNA.
 7. A transformant or a transductant, whichcomprises the recombinant DNA of the above
 6. 8. A method for producingglutaminase, which comprises culturing the transformant or transductantof the above 7 in a medium, and collecting glutaminase from the cultureproduct.